Monash Institute of Pharmaceutical Sciences (J.M.H., J.N.S., T.H.V., I.P.L.A., J.K.H., W.-L.L., K.L.) and Department of Anatomy and Developmental Biology (S.S., C.L.F., S.D.R.), Monash University, Victoria, Australia; and Department of Medicine, Royal Melbourne Hospital, Victoria, Australia (J.S.)
Monash Institute of Pharmaceutical Sciences (J.M.H., J.N.S., T.H.V., I.P.L.A., J.K.H., W.-L.L., K.L.) and Department of Anatomy and Developmental Biology (S.S., C.L.F., S.D.R.), Monash University, Victoria, Australia; and Department of Medicine, Royal Melbourne Hospital, Victoria, Australia (J.S.).
J Pharmacol Exp Ther. 2018 Nov;367(2):335-347. doi: 10.1124/jpet.118.250142. Epub 2018 Aug 13.
Renal podocyte survival depends upon the dynamic regulation of a complex cell architecture that links the glomerular basement membrane to integrins, ion channels, and receptors. Alport syndrome is a heritable chronic kidney disease where mutations in 3, 4, or 5 collagen genes promote podocyte death. In rodent models of renal failure, activation of the calcium-sensing receptor (CaSR) can protect podocytes from stress-related death. In this study, we assessed CaSR function in podocyte-like cells derived from induced-pluripotent stem cells from two patients with Alport Syndrome (AS1 & AS2) and a renal disease free individual [normal human mesangial cell (NHMC)], as well as a human immortalized podocyte-like (HIP) cell line. Extracellular calcium elicited concentration-dependent elevations of intracellular calcium in all podocyte-like cells. NHMC and HIP, but not AS1 or AS2 podocyte-like cells, also showed acute reductions in intracellular calcium prior to elevation. In NHMC podocyte-like cells this acute reduction was blocked by the large-conductance potassium channel (KCNMA1) inhibitors iberiotoxin (10 nM) and tetraethylammonium (5 mM), as well as the focal adhesion kinase inhibitor PF562271 (N-methyl-N-(3-((2-(2-oxo-2,3-dihydro-1H-indol-5-ylamino)-5-trifluoromethyl-pyrimidin-4-ylamino)-methyl)-pyridin-2-yl)-methanesulfonamide, 10 nM). Quantitative polymerase chain reaction (qPCR) and immunolabeling showed the presence of transcript and protein in all podocyte-like cells tested. Cultivation of AS1 podocytes on decellularized plates of NHMC podocyte-like cells partially restored acute reductions in intracellular calcium in response to extracellular calcium. We conclude that the AS patient-derived podocyte-like cells used in this study showed dysfunctional integrin signaling and potassium channel function, which may contribute to podocyte death seen in Alport syndrome.
足细胞的存活依赖于一种复杂的细胞结构的动态调节，这种结构将肾小球基底膜与整合素、离子通道和受体连接起来。Alport 综合征是一种遗传性慢性肾脏病，其中 3、4 或 5 型胶原基因突变促进足细胞死亡。在肾功能衰竭的啮齿动物模型中，钙敏感受体 (CaSR) 的激活可以保护足细胞免受应激相关死亡。在这项研究中，我们评估了来自 Alport 综合征（AS1 和 AS2）和无肾脏疾病个体[正常人系膜细胞（NHMC）]的诱导多能干细胞衍生的足细胞样细胞以及人永生化足细胞样（HIP）细胞系中的 CaSR 功能。细胞外钙引发所有足细胞样细胞中细胞内钙浓度依赖性升高。NHMC 和 HIP，但不是 AS1 或 AS2 足细胞样细胞，在升高之前也表现出细胞内钙的急性减少。在 NHMC 足细胞样细胞中，这种急性减少被大电导钾通道（KCNMA1）抑制剂 iberiotoxin（10 nM）和四乙铵（5 mM）以及粘着斑激酶抑制剂 PF562271（N-甲基-N-（3-（（2-（2-氧代-2,3-二氢-1H-吲哚-5-基氨基）-5-三氟甲基嘧啶-4-基氨基）-甲基）-吡啶-2-基）甲磺酰胺，10 nM）阻断。定量聚合酶链反应（qPCR）和免疫标记显示所有测试的足细胞样细胞中均存在 转录本和蛋白。AS1 足细胞在 NHMC 足细胞样细胞的脱细胞板上培养部分恢复了对细胞外钙的反应中细胞内钙的急性减少。我们得出结论，在这项研究中使用的 AS 患者衍生的足细胞样细胞显示出整合素信号和钾通道功能的功能障碍，这可能导致 Alport 综合征中观察到的足细胞死亡。
